WO2005033323A1 - Process for producing 1,4-dihydoxy-2-naphthoic acid - Google Patents

Abstract

A process for producing 1,4-dihydoxy-2-naphthoic acid, characterized by starting to culture 1,4-dihydoxy-2-naphthoic acid producing bacterium belonging to propionic acid bacteria under anaerobic conditions and when the concentration of carbon sources in the culture medium has become 3.5 mass% or below, conducting culturing while aerating the culture medium.

Description

 Specification

 Preparation of 1,4-dihydroxy-2-naphthoic acid

 Technical field

 The present invention relates to a method for producing 1,4-dihydroxy-2-naphthoic acid (hereinafter also referred to as DHNA) at a high concentration using fermentation of propionic acid bacteria, and a technique for improving the flavor of a culture thereof.

 Background art

 [0002] DHNA has conventionally been known to be useful as an industrial material as a dye, a pigment, and a photosensitive material, and various synthetic methods have been developed by organic chemical synthesis methods. The present inventors studied alternative DHNA production methods and found that DHNA was produced in large quantities in and out of the cells by propionic acid bacteria, and the DHNA-containing composition collected from this culture, or 1 , 4-Dihydroxy-2-naphthoic acid or its salt has the effect of reducing the abdominal discomfort seen when ingesting milk with lactose intolerance, and that it is useful for the prevention and treatment of metabolic bone disease (Patent Document 1). According to this method, it has been difficult to use DHNA-containing compositions, which make it possible to use DHNA in foods and drinks and pharmaceuticals, in products that are not always satisfactory in terms of flavor.

 Patent Document 1: International Publication No. WO03Z016544 pamphlet

 Disclosure of the invention

 Problems to be solved by the invention

[0003] It is an object of the present invention to provide a method for efficiently producing a DHNA-containing composition having improved flavor by propionic acid bacteria fermentation.

 Means for solving the problem

[0004] The present inventors have conducted intensive studies from various angles to obtain a DHNA-containing composition with reduced bitterness. As a result, the culture medium was air-ventilated at a certain time during fermentation of propionic acid bacteria. This led to a useful new finding that the concentration of DHNA in the culture was increased. It was also found that the DHNA concentration was increased by adding a carbon source of propionic acid bacteria to the culture after culturing and storing at low temperature under weak alkaline conditions, even though the culturing was completed. . In addition, the DHNA-containing composition obtained by brute force suppresses bitterness, It was found that the taste was good and that it was useful as a food or drink or pharmaceutical.

 That is, according to the present invention, the cultivation of 1,4-dihydroxy-2 naphthoic acid-producing bacteria belonging to the propionic acid bacterium was started under anaerobic conditions, and when the carbon source concentration in the medium became 3.5% by mass or less. An object of the present invention is to provide a method for producing 1,4-dihydroxy-2 naphthoic acid, which comprises culturing by aeration in a medium.

Also, the present invention provides a method for culturing a 1,4-dihydroxy-2 naphthoic acid-producing bacterium belonging to a propionic acid bacterium under anaerobic conditions, adding a carbon source to the obtained culture, and culturing the solution at a temperature of 3 to ²⁰ ° under a weak alkali. The present invention provides a method for producing 1,4-dihydroxy-2 naphthoic acid, which is stored in C.

 Further, the present invention provides a method for culturing a 1,4-dihydroxy-2 naphthoic acid-producing bacterium belonging to a propionic acid bacterium under an anaerobic condition, and when the carbon source concentration in the medium becomes 3.5% by mass or less, A method for producing 1,4-dihydroxy-2 naphthoic acid, characterized in that the culture is obtained by aeration, and a carbon source is added to the resulting culture and stored at 3-20 ° C under weak alkaline conditions. Is what you do.

 The present invention also provides a 1,4-dihydroxy-2 naphthoic acid-containing composition obtained as described above.

 Further, the present invention provides a food and beverage for improving abdominal discomfort symptoms, an agent for improving abdominal discomfort symptoms, an agent for preventing and treating metabolic bone diseases, which comprises the 1,4-dihydroxy-2 naphthoic acid-containing composition obtained as described above as an active ingredient. It is intended to provide a food or drink or an agent for preventing or treating metabolic bone disease.

 The present invention also relates to a food and drink for improving abdominal discomfort symptoms, an agent for improving abdominal discomfort symptoms, a food and drink for preventing and treating metabolic bone disease, or a metabolic food or drink, of the 1,4-dihydroxy-2 naphthoic acid-containing composition obtained as described above. The present invention also provides use of the composition for the prevention and treatment of bone diseases.The present invention also provides administration of an effective amount of the 1,4-dihydroxy-2 naphthoic acid-containing composition obtained as described above. It is intended to provide a method for treating abdominal discomfort or a method for treating metabolic bone disease.

The invention's effect [0006] According to the present invention, DHNA can be produced efficiently, and the obtained DHNA-containing composition has a good flavor and is useful as a food, beverage, or pharmaceutical.

 Brief Description of Drawings

 [0007] [Fig. 1] shows the change in DHNA concentration when the switching time to air rate is changed.

FIG. 2 shows the change in lactose concentration when the switching time to air rate is changed.

 FIG. 3 shows the timing of switching to the air rate and changes in the number of propionic acid bacteria.

 FIG. 4 shows the concentration of propionic acid when the switching time to the air rate is changed.

 FIG. 5 shows the acetic acid concentration when the switching time to the air rate is changed.

 FIG. 6 shows the DHNA concentration when the flow rate of the air rate was changed.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0008] The propionic acid bacterium used in the production method of the present invention is not particularly limited as long as it is a DHNA-producing bacterium, and a bacterium belonging to the genus Propio-bataterum is preferred. For example, propioninobacterium ' freudenreichii), Propio-bacterium 'Tenny (P. thoenii), Propio-batatherimu' P. acidipropionici (P. acidipropionici) ', Propio-batatherimu' P. jensenii ' Examples include butterium 'Avidum' (P. avidum), propio-bataterum 'Aknes (P. acnes), propio-batateurium' Linphofilum (P. lymphophilum), propio-bataterimu 'dala-urosum (P. granulosam) and the like. Of these, P. freudenreich ii IFO 12424, P. freudenreichii ATCC 6207, and P. freudenreichii ET-3 (FERM P-18454) are particularly preferred!

[0009] The medium used in the method of the present invention is preferably a medium containing a carbon source. In the present invention, the carbon source refers to a carbon source that can be assimilated by propionic acid bacteria. For example, lactose, glucose, lactic acid, glycerol, dartene, cellulose and the like can be mentioned, and lactose is particularly preferred. The content of the carbon source in the medium before the start of the culture is preferably 418% by mass, more preferably 417% by mass, and particularly preferably 417% by mass. Among these, a medium containing lactose as a carbon source includes whey powder, power zein, skim milk powder, or whey protein concentration obtained by dialyzing whey to reduce the lactose content. A whey protein isolate obtained by further separating the lactose content from the condensate or the lactose content may be mentioned. These can be used as they are or after being treated with protease, and can be used as a carbon source that can be used as a carbon source for assimilating peptone such as yeast extract and trypticase, and propionic acid bacteria such as glucose, lactose, and lactase-treated lactase. Animal and plant foods or extracts thereof containing a suitable amount of saccharides such as saccharides and Z or disaccharides, and minerals such as lactate, glycerol, dartene, cellulose, whey minerals, and, if necessary, oysters and ginger. The medium can be prepared by adding the medium. The following is an example of a method for preparing a medium containing a protease-treated skim milk powder as a medium material.

 [0010] The skim milk powder is dissolved in water so as to have a concentration of 10 to 20% by mass, and the temperature is adjusted to 47 ° C. To this, protease is added at 2.5% by mass of the skim milk powder to decompose the protein in the skim milk solution. Examples of the protease include proteolytic enzymes derived from animals and plants or bacteria, and can be used regardless of acidity, neutrality, or alkalinity. The digestion is performed for 6 hours, the temperature during the digestion is adjusted to 47 ° C and the pH is adjusted to 6.8. To adjust the pH, use a potassium carbonate aqueous solution. When the decomposition by the protease is completed, the skim milk solution is heated to 80 ° C. and kept for 10 minutes to inactivate the protease. After inactivation, mess up with water so that the mass concentration of skim milk powder becomes 10%, add brewer's yeast extract of 110 to 10 mass%, preferably 3 to 7 mass% of skim milk powder, and sterilize. . Sterilization conditions are 7 minutes or more at 121 ° C when using an autoclave, and 4 seconds or more at 140 ° C or more when a sterile plate is used. The culture medium thus obtained usually contains lactose in an amount of 45% by mass.

[0011] The culture is performed under anaerobic conditions. The anaerobic condition can be, for example, one or a combination of two or more of nitrogen gas, helium gas, argon gas, hydrogen gas, and other inert gas. . More specifically, nitrogen gas, carbon dioxide gas, etc. are flowed through the top of the fermenter by aeration on the top surface, stirring is performed, and the medium temperature is adjusted to 33 ° C. When the medium temperature is stabilized at 33 ° C, inoculate a starter for propionic acid bacteria and start cultivation under anaerobic conditions. As the starter, an activated culture solution of propionic acid bacteria, a cell concentrate of the culture solution, and the like can be used. The amount to be added to the medium is about 0.05% of the medium in the former case, and about 0.3% in the latter case as a guideline.These amounts can be changed even if necessary as needed. . [0012] Culture is performed under the conditions of a culture temperature of 20 to 40 ° C and a medium pH of neutral to slightly acidic (preferably pH 5.5 to 7.5). In order to suppress the increase in the acidity during the culture, a known neutralizing agent such as an aqueous solution of potassium carbonate or an aqueous solution of sodium carbonate can be used.

Next, a method of aerating the culture medium will be described. It is surprising that this measure increases DHNA production. It is not clear why the continuous aeration increases DHNA production, but this aeration causes propionic acid bacteria to begin consuming propionic acid.

[0014] The time when the air rate is started is when the carbon source concentration in the culture medium becomes 3.5% by mass or less. One guideline is 24 hours before the carbon source of the propionic acid bacterium dies. You. It is more preferable to start the aeration when the carbon source concentration reaches 1.0 to 3.5% by mass, particularly 1.5 to 3.0% by mass. By aeration when the carbon source concentration falls below 3.5% by mass, propionic acid bacteria also consume cacaine and propionic acid as a carbon source, and eventually the carbon source almost depletes. . The number of propionic acid bacteria in the culture medium at the start of aeration should be 1.OX 10 ^1G cfu / mL (10.0 log cfu / mL) or more, and 1.4 X 10 ^1G cfuZmL (10.1 log cfuZmL) or more. preferable. By doing so, the carbon source in the medium can be almost depleted. In the above-mentioned lactose-containing medium and culture conditions, about 48 hours after the start of the culture, the lactose concentration becomes 3.5% by mass or less, and it is time to start the aeration. In addition, a culturing method in which saccharides such as lactose and glucose serving as carbon sources of propionic acid bacteria are added in the middle is known (for example, Patent Document 1, Japanese Patent Application Laid-Open No. 10-304871), but in the present invention, these saccharides are used. It is preferable to let the syrup be dried without adding the soybean curd on the way.

[0015] The amount of air supplied by the air rate is preferably an amount that stimulates propionic acid bacteria. As an example corresponding to this condition, a specific example on a laboratory scale (1.5 L capacity) is as follows. When culturing at 150 rpm with a stirring panel using a sparger, the air supply rate is 2 L or more for Z minutes. The amount is preferably 2 LZ minutes to 4 LZ minutes, but can be appropriately adjusted according to the capacity, stirring speed, apparatus and the like. If the amount of dissolved oxygen in the solution becomes unnecessarily high, growth of propionic acid bacteria is stopped, and DHNA production is stopped. Round. In the case of the above-mentioned medium and culture conditions, the culture is usually completed in about 168 hours from the start of the culture.

 [0016] Examples of the method of aeration include a method of inserting a porous ventilation tube into a culture medium and sending air over the entire surface of the tube, and a method of sending bubbles using a sparger.

[0017] In this way, the DHNA accumulated in the medium and the cells can be stopped for culturing and immediately used for DHNA collection from the culture. In addition, the end point of the culture is about 3 to 5 days when the number of bacteria reaches the stationary phase and the carbon source in the medium dies.

 Next, a method for producing DHNA by adding a carbon source of propionibacteria to a culture after completion of the culture and storing the culture at 321 ° C. under a weak alkali will be described. Here, the culture may be the culture after the above-described aeration, or may be the culture after the completion of the culture under ordinary anaerobic or microaerobic conditions without passing through the aeration.

[0019] The amount of the carbon source added to the culture is such that the carbon source concentration in the culture is 0.2 to 3.0% by mass, more preferably 0.4 to 2.5% by mass, particularly 0.8 to 2%. It is preferable to set the amount to 2% by mass. To make the culture weakly alkaline, add a base such as potassium carbonate, sodium carbonate or sodium phosphate to adjust the pH of the culture to 7-9, especially 7.5-8.5. Is preferred. Further, the storage temperature is 3-20 ° C, particularly 3-15 ° C, and more preferably 5-15 ° C. The storage period is preferably 1 to 3 weeks, particularly preferably 1 to 2 weeks.

[0020] Such low-temperature storage under a weak alkali improves the DHNA content in the culture. Thus, this method, which does not require new equipment, saves space, and can increase the amount of DHNA during storage, is extremely useful and effective!

Next, a method of collecting DHNA will be described. The obtained culture is preferably subjected to adsorption chromatography. As the adsorbent, a reversed-phase adsorbent such as activated carbon or a synthetic adsorbent (for example, Diaion HP-20, manufactured by Mitsubishi Iridaku Co., Ltd.) can be widely used. First, the column is filled with the adsorbent and washed with 0.5% (wZv) sodium ascorbate aqueous solution. Next, the obtained culture is added to a column (the passing solution is referred to as "pass"), and the water-soluble fraction is removed with a 0.5% (wZv) aqueous sodium ascorbate solution. After that, 0.5% (w / v) amount of sodium ascorbate was eluted with ethanol added with sodium chloride and this ethanol was added. By concentrating the eluted fraction, a composition containing DHNA at a high concentration can be obtained. Furthermore, purification can be performed to obtain pure DHNA or a salt thereof. Note that another alcohol such as methanol may be used instead of ethanol as the eluate of DHNA from the column. As an alternative, DHNA can be separated from the supernatant obtained by centrifuging the culture and collecting the DHNA using liquid chromatography. In addition, it is also preferable to use sodium erythorbic acid instead of sodium ascorbate. These are useful as stabilizers for DHNA. In the present invention, ascorbic acid, erythorbic acid, and those free acids are mainly used.ゝ, fatty acid esters and other various esters, alkali metal salts and other salts can also be used.

 [0022] Examples of DHNA salts include pharmaceutically or food acceptable salts. Representative salts include monovalent metal salts such as sodium, potassium, and lithium, and magnesium, calcium, and zinc. Examples thereof include polyvalent metal salts, and inorganic or organic amine salts such as ammonia and ethanolamine. In addition, salt exchange can also be performed by using a reaction known per se. Examples of the salt include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citrate) And malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid), but these are only examples, and the present invention is not limited to these salts.

 [0023] Since DHNA is contained in the culture of the DHNA-producing bacteria (inside and inside or outside of the cells), the culture itself is used without application of adsorption chromatography using a rotary evaporator or the like. By concentrating, a composition containing a high concentration of DHNA can be obtained. It is also preferable to separate the cells from the culture by a usual centrifugation method and concentrate the supernatant. The composition thus obtained may be used in a liquid state or may be processed into a powder according to the form to be used.

[0024] The DHNA-containing composition obtained by force exerts a high DHNA concentration, suppresses bitterness, and has a good flavor. Therefore, the DHNA-containing composition of the present invention, or DHNA or a salt thereof, can be used in the form of a food or drink or a medicament, for example, by directly administering it as a medicament, or special food such as food for specified health use. By ingesting directly as intended foods or functional foods, or by adding various foods (milk, soft drinks, fermented milk, 1 dart, cheese, bread, biscuit, cracker, pizza crust, etc.), and ingesting it can improve intestinal flora and reduce abdominal discomfort seen when consuming milk. It can prevent and treat metabolic bone disease.

 [0025] In order to produce the food, water, protein, saccharide, lipid, vitamin, and minerals, organic acid, fruit juice, flavor, and the like can be combined as main components. For example, whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, β-latatoglobulin, and lactalbumin Animal and plant proteins such as ratatoferrin, soy protein, chicken egg protein, meat protein, etc., and their various hydrolysates, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose, etc. Ingredients: sucrose, glucose, fructose, sugar alcohols, maltose, oligosaccharides, modified starch (such as dextrin, soluble starch, pretty starch, oxidized starch, starch ester, starch ether, etc.), and carbohydrates such as dietary fiber; Animal oils such as fish oil; palm oil, safflower oil, corn oil, Vegetable oils and fats such as seed oil and coconut oil, their fractionated oils, hydrogenated oils, and vegetable oils such as transesterified oils; vitamin Α, vitamin Β, vitamin C, erythorbic acid, vitamin D, vitamin E, Various vitamins such as vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, piotin, inosit, choline, folic acid; calcium, potassium, magnesium, sodium, chlorine, copper, iron, manganese, zinc, Minerals such as selenium, fluorine, silicon, and iodine; organic acids and organic acid salts such as malic acid, citric acid, lactic acid, and tartaric acid; and one or more selected from these as appropriate. Can be added. It is desirable to add these various components to foods containing a large amount of these components as well as synthetic products. The form is not limited to the above-mentioned foods, and is not particularly limited as long as the activity is maintained in the final product. Liquid, solid (including granules, powders, tablets, and gels) and semi-solids It may be in any form (including jelly), paste, emulsified, and the like.

When the composition according to the present invention or DHNA or a salt thereof is used as a pharmaceutical, it can be administered in various forms. Examples of the form include oral administration by tablets, capsules, granules, powders, syrups and the like. These various products The agent can be used in the pharmaceutical formulation technical field such as excipients, binders, disintegrants, lubricants, flavors, flavoring agents, solubilizers, suspending agents, coating agents, etc. in the main agent according to the usual method. It can be formulated using known auxiliaries.

 Example

 Hereinafter, the present invention will be described with reference to Test Examples and Examples, but the present invention is not limited thereto. In the following Test Examples and Examples, the quantification of DHNA was performed according to the method described on page 9 of WO03Z016544. The lactose concentration was measured by a flow injection analysis method using a lactose electrode (using a flow injection analyzer, Bio Flow Analyzer (trade name) manufactured by Oji Scientific Instruments). The number of propionic acid bacteria was measured on a BL agar medium. The concentrations of propionic acid and acetic acid were measured by an HPLC method (column: RS pa k KC-811 + pre-column KCG, detection: UV445 nm).

 [Test Example 1] Examination of air-rate switching timing

 (1) Preparation of medium

 [0028] 150 g of skim milk powder (manufactured by Meiji Dairies Co., Ltd.) was dissolved in 100 g of water, and the temperature was adjusted to 47 ° C.

 To this, 3.75 g of protease was added, and protein degradation was performed at 47 ° C for 6 hours. The pH during protein degradation was adjusted to 6.6-6.8 using potassium carbonate solution. After proteolysis, the protease was inactivated by heating to 80 ° C and holding for 10 minutes, 7.5 g of brewer's yeast extract was added, and the pH was adjusted to 6.95 using an aqueous potassium carbonate solution. The volume of the solution was adjusted to 1500 mL with water, and the solution was placed in a 2 L fermenter to sterilize the medium. Sterilization conditions were 121 ° C for 7 minutes.

 (2) Culture conditions

 [0029] Nitrogen gas was passed through the fermenter, and the temperature of the medium was stabilized at 33 ° C. Then, 0.75 mL of a freeze concentration starter (P. freudenreichii ET-3) was added, and culture was started. During fermentation, the temperature was adjusted to 33 ° C, the pH was adjusted to 6.5, and nitrogen gas was aerated. The pH was adjusted using a 40% (wtZ wt) aqueous potassium carbonate solution. The following five types of culture methods were implemented.

1) Continue nitrogen gas aeration from the start to the end of the culture,

2) As in 1), except that lactose 2% by weight of the culture solution was added 72 hours and 96 hours after the start of culture. 3) Twenty-four hours after the start of the culture, switch the nitrogen gas aeration to air rate (for 2 LZ),

4) After 48 hours, the cultivation starting power was switched to air rate (2LZ).

 5) After 72 hours from the start of the culture, switch to the air rate (for 2LZ),

 All of these were terminated after 168 hours from the start of the culture.

 (3) Results

[0030] Time-dependent changes in DHNA concentration, lactose concentration, the number of propionic acid bacteria, propionic acid concentration, and acetic acid concentration are shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, and Fig. 5, respectively. As is clear from these results, the culture obtained in 4) and 5) had a DHNA concentration of about 45 / z gZmL (FIG. 1). In 4) and 5), it was confirmed that lactose almost died after 96 hours (Fig. 2), and the propionic acid concentration, which had also increased the cultivation starting power, gradually decreased (Fig. 4). Except for 3), the number of propionic acid bacteria exceeded 1.OX 10 ¹⁰ cfu / mL (10.0 log cfu / mL) at the end of culture, and 4) and 5) were almost 1.0 X 10 cfu / mL (11. 0 log cfu / mL) was reached (Fig. 3).

 [0031] From the above, it was confirmed that a culture containing a high concentration of DHNA could be obtained by switching to nitrogen aeration aeration after at least 48 hours of the culture start force (Fig. 6). The lactose concentration at the time of switching was 3.3% by mass in 4) and 2.9% by mass in 5) (Fig. 2).

 [Test Example 2] Examination of air rate

[0032] Culture was performed under the same conditions as in 5) of Test Example 1 except that the amount of aeration was changed. After 72 hours from the start of fermentation, the culture was carried out with the air flow rate changed to 0.5 LZ, 1.0 LZ, 2.0 LZ, and 4.0 LZ. As a result, it was clarified that the DHNA concentration became about 40 gZmL 144 hours after the start of culture by setting the flow rate to 2.0Z minutes or more.

 [Example 1]

 180 g of skim milk powder (manufactured by Meiji Dairies Co., Ltd.) was dissolved in 100 g of water, and the temperature was adjusted to 47 ° C.

The protein was decomposed at 47 ° C for 3 hours after adding 3.75g of protease to the mixture. During protein degradation, ρ 分解 was adjusted using an aqueous potassium carbonate solution at 6.6-6.8. After proteolysis, the protease was inactivated by heating to 80 ° C and holding for 10 minutes, 7.5 g of beer yeast extract and 15 g of lactose were added, and the pH was adjusted to 6.95 using an aqueous potassium carbonate solution. did. The volume of the solution was adjusted to 1500 mL with water, and the solution was placed in a 2 L fermenter, and the medium was sterilized (lactose concentration mass: about 6.1%). Sterilization conditions were 121 ° C for 7 minutes. After sterilization, nitrogen gas was passed through the fermenter, the medium temperature was stabilized at 33 ° C, and 0.75 mL of freeze-concentration starter (P. freudenreichii ET-3) was added to the culture, and the culture was started. . During the culture, the temperature was adjusted to 33 ° C, the pH was adjusted to 6.5, and nitrogen gas was passed. To adjust the pH, a 40% (wtZ wt) aqueous solution of potassium carbonate was used. The culture was switched to nitrogen gas ventilation 72 hours after the start of the culture, and the culture was terminated 168 hours after the start of the culture. The air flow rate during the air rate was 2 LZ, and the stirring speed was 150 rpm. As a result, a culture having a DHNA concentration of 52 gZmL was obtained. After 72 hours, the lactose concentration was about 1.9% by mass, and the number of propionic acid bacteria was 3.5 ^× 10 ^lc> cfu / mL.

 [Example 2]

[0034] 120 kg of skim milk powder (manufactured by Meiji Dairies Co., Ltd.) was dissolved in 750 kg of water, and the temperature was adjusted to 47 ° C. To this, 2.5 kg of protease was added, the pH was adjusted to 7.6, and protein degradation was performed at 47 ° C for 3 hours. After the decomposition was completed, the protease was inactivated by heating to 80 ° C and holding for 10 minutes, 5 kg of brewer's yeast extract and 10 kg of lactose were added, and the medium was sterilized at 140 ° C for 4 seconds. The medium pH before the start of sterilization was 6.9. After sterilization, adjust the volume of the medium to 1000 L with water (lactose concentration approx. 6.1% by mass), inject nitrogen gas into the fermenter for 20 LZ, stabilize the medium temperature at 33 ° C, and start 3.0 L of freudenreichii ET-3) was added. During fermentation, the temperature was adjusted to 33 ° C, the pH was adjusted to 6.5, and nitrogen gas was passed. The pH was adjusted using a 23% (wt / wt) aqueous potassium carbonate solution. At 72 hours after the start of the culture, the gas permeability of the nitrogen gas was also switched to the air rate, and the culture was terminated at 168 hours after the start of the culture. The air flow rate during the air rate was 200 LZ, and the stirring speed was 52 rpm. As a result, a culture having a DHNA concentration of 42 gZmL was obtained. The lactose concentration 72 hours after the start of the culture was about 1.5% by mass, and the number of propionic acid bacteria was 3. OX10 ^1G cfu / mL.

 [Example 3]

[0035] The DHNA-containing culture obtained in Example 2 was supplemented with sodium ascorbate (1.0%) and lactose (2.0%), and the pH was adjusted to 8.0. As a result of storage at ° C for 2 weeks, the DHNA concentration was 55 µgZmL. When lactose was replaced with glucose and stored in the same manner, the DHNA concentration increased from the end of the culture.

 [Comparative example]

 [0036] During the cultivation, all the operations were performed under the same conditions as in Example 1 except that the nitrogen gas flow was continued without switching to the air rate. As a result, a culture having a DHNA concentration of 32 gZmL was obtained.

 [Example 4]

 [0037] The DHNA-containing culture obtained in Example 1 was added to 120 g of plain yogurt (manufactured by Meiji Dairies Co., Ltd.), and the sensory evaluation of the prepared yogurt is shown in Tables 1 and 2. The yogurt to which the DHNA-containing culture obtained by the method of the present invention was added had a sour taste as compared with the conventional method in which the DHNA concentration was high (the DHNA-containing culture obtained in Comparative Example was added to plain yogurt). It was confirmed that no bitterness was felt.

 [0038] [Table 1] Item Flavor Sourness Bitterness Overall evaluation

 © 調製 prepared in Example 1-©

 Culture

 〇 〇 X mm prepared in Comparative Example

 Culture

 X: Bad Mu: Somewhat bad 〇: Normal ◎ Excellent

 Add 1 g to plain yogurt

 Plain yogurt: 120g

[Table 2] Item Flavor Sourness Bitterness Overall evaluation

 Δ 〇-調製 prepared in Example 1

 Culture

 Δ Δ X X prepared in Comparative Example

 Culture

 X: Poor △: Slightly poor 〇: Normal ◎ Excellent I: Not felt

 Add 2g to plain yogurt

 Plain yogurt: 120g

[Example 5]

[0041] 120 kg of skim milk powder (manufactured by Meiji Dairies Co., Ltd.) was dissolved in 750 kg of water, and the temperature was adjusted to 47 ° C. . To this, 2.5 kg of protease was added, the pH was adjusted to 7.6, and protein degradation was performed at 47 ° C for 6 hours. After the decomposition was completed, the protease was inactivated by heating to 80 ° C and holding for 10 minutes, 5 kg of brewer's yeast extract and 10 kg of lactose were added, and the medium was sterilized at 140 ° C for 4 seconds. The medium pH before the start of sterilization was 6.9. After sterilization, adjust the volume of the medium to 1000 L with water (lactose concentration approx. 6.1% by mass), inject nitrogen gas into the fermenter for 20 LZ, stabilize the medium temperature at 33 ° C, and start 3.0 L of freudenreichii ET-3) was added. During the culture, the temperature was adjusted to 33 ° C, the pH was adjusted to 6.5, and nitrogen gas was passed. The pH was adjusted using a 23% (wt / wt) aqueous potassium carbonate solution. At 72 hours after the start of the culture, the gas permeability of the nitrogen gas was also switched to the air rate, and the culture was terminated at 168 hours after the start of the culture. The air flow rate during the air rate was 200 LZ, and the stirring speed was 52 rpm. As a result, a culture having a DHNA concentration of 48 gZmL was obtained. The lactose concentration 72 hours after the start of the culture was about 3.0% by mass, and the number of propionic acid bacteria was ^2.9 × 10 ^{1 G} cfu / mL.

[Example 6]

 The DHNA-containing culture obtained in Example 5 was added with 1.0% sodium ascorbate and 1.0% lactose to adjust the pH to 8.0, and then adjusted to 10 ° C. As a result of storage for 2 weeks, the DHNA concentration was 60 μg ZmL.

Claims

The scope of the claims  [I] Culture of 1,4-dihydroxy-2-naphthoic acid-producing bacteria belonging to the propionic acid bacterium was started under anaerobic conditions, and when the carbon source concentration in the medium became 3.5% by mass or less, the medium was added to the medium. A method for producing 1,4-dihydroxy-2-naphthoic acid, which comprises culturing in an array.  [2] The method according to claim 1, wherein the medium is a medium containing 418% by mass of a carbon source. 3. The method according to claim 1, wherein the anaerobic condition is a condition in a nitrogen gas or carbon dioxide gas atmosphere.  [4] A 1,4-dihydroxy-2-naphthoic acid-producing bacterium belonging to the propionic acid bacterium is cultured under anaerobic conditions, and a carbon source is added to the resulting culture, which is stored at 3-20 ° C under a weak alkali. A process for producing 1,4-dihydroxy-2-naphthoic acid, which comprises:  5. The production method according to claim 4, wherein the amount of the carbon source added to the culture is such that the carbon source concentration in the culture becomes 0.2 to 3% by mass.  [6] The production method according to claim 4 or 5, wherein the storage is performed by setting the pH of the culture to 7 to 9 and storing the culture at 3 to 20 ° C for one to three weeks.  [7] The culture of 1,4-dihydroxy-2-naphthoic acid-producing bacteria belonging to the propionic acid bacterium was started under anaerobic conditions, and when the carbon source concentration in the medium became 3.5% by mass or less, the medium was added to the medium. A method for producing 1,4-dihydroxy-2-naphthoic acid, comprising culturing in an array, adding a carbon source to the obtained culture, and storing the resulting culture at 320 ° C. under weak alkaline conditions.  [8] The method according to claim 7, wherein the medium is a medium containing 4.8% by mass of a carbon source.  9. The method according to claim 7, wherein the anaerobic condition is a condition in a nitrogen gas or carbon dioxide gas atmosphere.  10. The method according to claim 7, wherein the amount of the carbon source added to the culture is such that the concentration of the carbon source in the culture is 0.2 to 3% by mass.  [II] The method according to any one of claims 7 to 9, wherein the storage is performed by keeping the culture at a pH of 7 to 9 and storing at 3 to 20 ° C for one to three weeks. [12] A 1,4-dihydroxy-2-naphthoic acid-containing composition obtained by the production method according to any one of claims 11 to 11. [13] A food or drink for improving abdominal discomfort, comprising the composition according to claim 12 as an active ingredient.  [14] An agent for improving abdominal discomfort, comprising the composition according to claim 12 as an active ingredient.  [15] A drink for preventing or treating metabolic bone disease, comprising the composition according to claim 12 as an active ingredient.  TOo  [16] A prophylactic / therapeutic agent for a metabolic bone disease, comprising the composition according to claim 12 as an active ingredient. [17] Use of the composition according to claim 12, for producing a food or drink for improving abdominal discomfort. [18] Use of the composition according to claim 12, for producing an agent for improving abdominal discomfort symptoms. [19] Use of the composition according to claim 12 for producing a food or drink for preventing or treating metabolic bone disease. [20] Use of the composition according to claim 12 for producing an agent for preventing or treating a metabolic bone disease. [21] A method for treating abdominal discomfort, which comprises administering an effective amount of the composition according to claim 12.  [22] A method for treating a metabolic bone disease, which comprises administering an effective amount of the composition according to claim 12.